This project is aimed at the interactions between neurofilaments, microtubules and their associated proteins in vitro using a variety of biochemical techniques. A divalent ion activated, EDTA-inhibited ATPase associated with both microtubules and neurofilaments has been identified in brain and will be purified by subfractionation and by use of intact microtubules as affinity probes. Studies on the development, synthesis and turnover of neurofilaments will be carried out in clonal PC12 pheochromocytoma cells, in cultured sympathetic neurons and in the neonatal rat optic nerve. Attempts will be made to establish models of neurofibrillary proliferation in pure neuronal cultures and to determine the mechanism of formation of the filamentous bundles. A new, non-denaturing purification for soluble glial filament subunits will be developed. The purified protein will be used to study the assembly and disassembly conditions for this protein in vitro. Work on the purification of paired helical filaments from Alzheimer's Disease will continue. Antibodies prepared to the 25 kd protein of neurofilaments will be tested for activity against PHF-containing neurons.